In all mammalian species examined, there is a 10 mV or more gradient in resting membrane potential (RMP) across the muscle wall of the gastric antrum, small intestine and large intestine, and an even larger gradient along the long axis of the stomach. These voltage gradients, which may be considered biological rheostats, are central to the ability of circular smooth muscle to vary the strength of contraction from weak to propulsive and occluding. Our previous work shows that constitutive heme oxygenase 2 (HO-2), the enzyme that generates carbon monoxide (CO), is present in interstitial cells of Cajal (ICC) and that exogenous CO hyperpolarizes the membrane potential freshly dissociated circular smooth muscle cells. We hypothesize that CO is generated in different amounts across the thickness of the gut wall with more generated in regions where RMPs are more hyperpolarized compared to regions where RMPs are more depolarized. We focus on CO generated in ICC because RMPs in circular smooth muscle cells are significantly depolarized when ICC is absent or when HO2 is absent (HO-2-/- mice) but are normal in mice lacking an enteric nervous system. Our experiments are designed to test the hypothesis that CO is generated in the gut wall and that it acts on circular smooth muscle cells to hyperpolarize the RMP. Our work is organized under six Specific Aims. We began in Specific Aim I by measuring RMPs across the circular smooth muscle layer throughout the gastrointestinal tract of the human, canine and murine species and end with Specific Aim 6 designed to "rescue" primary cultures of depolarized smooth muscle cells by adding CO-producing ICC to the culture. In between these two aims, we will directly measure CO generation in hyperpolarized and depolarized regions of the gut wall, measure CO production from enriched ICC cell cultures, measure bilirubin production as marker of HO activity, and use Laser Capture Microdissection to selectively obtain distinct populations of cells of analysis of HO-2 copy number by microarrays and real time PCR. The significance of our work is two fold. Firstly, it will provide data to support the notion that CO generated in the gut wall is a hyperpolarizing factor and the root of the essential voltage gradient across the gut wall. Secondly, it may be of more generalized physiologic significance. Our work on CO in the gut may lend support to the general hypothesis that endogenously generated CO in smooth muscle tissues is a central factor in maintaining normal physiological function.